0090-9556/05/3304-596–602$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics
DMD 33:596–602, 2005

Vol. 33, No. 42782/1197647
Printed in U.S.A.

BUCOLOME, A POTENT BINDING INHIBITOR FOR FUROSEMIDE, ALTERS THE
PHARMACOKINETICS AND DIURETIC EFFECT OF FUROSEMIDE: POTENTIAL FOR USE
OF BUCOLOME TO RESTORE DIURETIC RESPONSE IN NEPHROTIC SYNDROME
Norito Takamura,1 Toru Maruyama, Etsuo Chosa, Keiichi Kawai, Yasuhiro Tsutsumi,
Yukie Uryu, Keishi Yamasaki, Tsuneo Deguchi, and Masaki Otagiri
Department of Pharmacy, Miyazaki Medical College Hospital, Miyazaki, Japan (N.T., K.Y.); Department of Biopharmaceutics,
Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan (T.M., Y.T., Y.U., T.D., M.O.);
Department of Orthopedic Surgery, Miyazaki Medical College, Miyazaki, Japan (E.C.); and School of Health Sciences, Facultyof Medical Kanazawa University, Kanazawa, Japan (K.K.)
Received October 29, 2004; accepted January 4, 2005

To determine whether bucolome (5-n-butyl-1-cyclohexyl-2,4,6-trioxoperhydropyrimidine), a nonsteroidal anti-inflammatory agent,can reverse diuretic resistance of furosemide in patients with nephrotic syndrome, we examined the inhibitory effect of bucolomeon the protein binding of furosemide in serum and urine. Bucolome
significantly inhibited the protein binding of furosemide not only inserum but also in urine of preparation albumin (UPA), which mimics
urinary albumin concentration in patients with nephrotic syndromeby ultrafiltration method. The binding percentage of furosemide toalbumin was approximately 70% in UPA. With coadministration of
bucolome to healthy volunteers, renal clearance of furosemide
was increased, reflecting the increase of the free fraction of furosemide in serum. Furthermore, coadministration of bucolome

caused a significant increase of urine volume and sodium concentration in urine. Even at higher urine levels of furosemide, theinhibitory effect of bucolome on the protein binding of furosemide
in UPA remains constant, and changes in pH at weakly acidic pH
levels (pH 5.5–6.5) did not alter the inhibitory effect of bucolome.
Interestingly, coadministration of bucolome with furosemide indoxorubicin (Adriamycin)-induced nephrotic syndrome model rats
alleviated the diuretic resistance. These results suggest that bucolome has a potent inhibitory effect on the protein binding of
furosemide in the urine and can partially restore the diuretic response of furosemide in patients with nephrotic syndrome by increasing the free fraction of furosemide at the site of action.

Attenuated response to diuretics is frequently observed in patientswith nephrotic syndrome (Green and Mirkin, 1980; Keller et al., 1982;
Smith et al., 1985; Kirchner et al., 1990, 1992; Wilcox, 2002).
Dose-response analysis comparing urinary excretion rates of diuretic
and sodium has demonstrated that nephrotic patients are less responsive to a given dose of drug than healthy subjects (Smith et al., 1985).
It has been proposed that this blunted response is due to tubular
resistance to the natriuretic effect of loop diuretics. Another possible
contribution to diuretic resistance in patients with nephrotic syndrome
is suggested by the finding that furosemide readily binds to albuminin serum (Takamura et al., 1996, 1997, 1998), which implies that it
also binds to albumin in urine. Since the protein-bound fraction of adrug is considered to be inactive (Martin, 1965; Meyer and Guttman,
1968; Vallner, 1977), binding of furosemide to urinary albumin would
decrease the effective drug concentration by reducing its unboundfraction at its site of action in the thick ascending limb of Henle’s

loop. This suggests that binding inhibitors for furosemide can normalize the natriuretic effect of the drug in renal tubules.
To have a significant renal effect, a protectable inhibitor shouldhave the following properties: 1) it should be a potent inhibitor of theprotein binding of furosemide in urine; 2) when administered in large
doses, its plasma concentration should reach high levels; 3) it should
be excreted mainly in urine; and 4) it should be highly safe andsuitable for repeated administration.
Kirchner et al. (1991) reported that binding inhibitors such as
warfarin and sulfisoxazole alleviated the blunted response to furosemide in rats in their in vivo loop microperfusion experiments.
However, warfarin is eliminated by metabolism rather than urinary
excretion, despite it has high affinity for albumin (Kragh-Hansen,
1981; Maes et al., 1982). In addition, warfarin can only be administered in small doses. Some sulfa drugs, including sulfisoxazole andsulfamethizole, are eliminated mainly by urinary excretion but have
low affinity for albumin (binding affinity, nK ⫽ 1 ⫻ 104 M⫺1 to 5 ⫻104 M⫺1) (Vallner, 1977; Kragh-Hansen, 1981), suggesting that thesedrugs would not be effective in restoring the blunted response to
furosemide in patients with nephrotic syndrome (Agarwal et al.,2000).
Previously, we reported that furosemide binds to site I on human1
Current address: Second Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, Yoshino,
Nobeoka, Japan.
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.104.002782.

ABBREVIATIONS: HSA, human serum albumin; UPA, urine of preparation albumin; RSA, rat serum albumin; NS rat, nephrotic syndrome model
rat; HPLC, high-performance liquid chromatography; CLtot, total body clearance; CLr, renal clearance; AUC, area under the curve.
596

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ABSTRACT:597

BUCOLOME ADVANCES THE DIURETIC EFFECT OF FUROSEMIDE

furosemide. In separate experiments, free furosemide concentration in humans
was determined by ultrafiltration techniques.
The concentration of furosemide in serum was determined using the HPLC
system described above. An Inertsil ODS column (5 ␮m) (GL Sciences Inc.,Tokyo, Japan) was used as the stationary phase. A 0.2-␮g aliquot containing
the internal standard, ethyl p-hydroxybenzoate, was added to 0.1-ml serum
samples. This mixture was vortexed, followed by addition of 0.5 ml of 1 M
potassium dihydrogenphosphate and 3 ml of diethyl ether. The mixture wasshaken for 10 min and then centrifuged (3000 rpm) for 10 min. The upper layer(2 ml) was transferred to a clean test tube and evaporated under nitrogen todryness. Then, 250 ␮l of mobile phase was added to the tube, and the resultant
mixture was injected into the HPLC apparatus. HPLC was performed at a flowrate of 1.1 ml/min at 40°C with a column oven, using 50 mM potassium
dihydrogenphosphate/acetonitrile (68:32 v/v) as the mobile phase for furosemide (retention time, 8.0 min). A UV monitor was used to assay for
furosemide and ethyl p-hydroxybenzoate, at a UV wavelength of 270 nm forboth.
Analysis of In Vitro Binding of Furosemide. Binding parameters were
estimated by fitting the experimental data to the following equation using anonlinear least-squares computer program (MULTI) (Yamaoka et al., 1981):

Materials and Methods
Materials. Furosemide powder and furosemide intravenous solution (20mg/ml) were donated by Hoechst Japan Co. (Tokyo, Japan). Adriamycin was
donated by Kyowa Hakko Kogyo Co. (Tokyo, Japan). HSA (essentially fatty
acid-free) and rat serum albumin (RSA) were purchased from Sigma-Aldrich(St. Louis, MO). RSA was defatted with activated charcoal in solution at 0°C,
acidified with H2SO4 to pH 3, and then freeze-dried (Tsutsumi et al., 1999).
Bucolome (5-n-butyl-1-cyclohexyl-2,4,6-trioxoperhydropyrimidine; Grelan
Pharmaceutical Co. Ltd., Tokyo, Japan) was obtained as a pure substance fromthe manufacturer. All other chemicals were of analytical grade.
Protein Binding Study. Ultrafiltration experiments were performed using a
Tosoh plastic ultrafiltration apparatus (Tosoh, Kanagawa, Japan). Aliquots (0.9
or 1.35 ml) of different concentrations of furosemide and a constant concentration of HSA (in 63 mM sodium phosphate buffer, pH 7.4), serum protein,
RSA, or urine of preparation albumin (UPA), which mimics urinary albumin
concentration observed in patients with nephrotic syndrome by adding the
HSA to urine of healthy volunteers, were centrifuged at 1200 or 2200g for 15
min at 25°C. Adsorption of drugs onto the membrane or apparatus was
negligible. No protein leakage was detected during the experiment.
The free concentrations of furosemide and bucolome were determined by
HPLC using a system consisting of a Shimadzu LC-6A UV detector (Shimadzu, Kyoto, Japan) and a Shimadzu SPD-6A pump. For both drugs, a
Superspher 100 RP-18e column (Cica Merck, Tokyo, Japan) was used as thestationary phase. Both furosemide (retention time, 4.3 min) and bucolome(retention time, 5.3 min) were assayed by UV monitoring at 280 nm. HPLC
was performed at a flow rate of 1.0 ml/min at 40°C with a column oven, using
distilled water/acetonitrile/methanol (491:9:4 v/v), and distilled water/acetonitrile (119:6 v/v) as the mobile phase for furosemide and bucolome,
respectively.
Effect of Bucolome on the Natriuretic Effect of Furosemide in Healthy
Volunteers. The protocol was approved in advance by the Ethical Committeeof Miyazaki College Hospital. A family member of the volunteer was asked togive informed consent when the volunteer was unable to do so, according toJapanese legislation. The subjects were three normal male humans, 34 to 48
years old and weighing 65 to 79 kg, who participated as outpatients. Eachsubject had a normal medical history, physical condition, and standard laboratory test results. Intake of sodium and fluid was maintained at constant levels
in all experiments. It has been reported that the tmax of bucolome is 4.5 h inhumans (Yashiki et al., 1971b). To obtain the maximum displacement effect by
bucolome for the protein biding of furosemide, each subject was intravenously
administered 20 mg of furosemide, alone or 4.5 h after pretreatment withbucolome (600 mg p.o.). An interval of at least 2 weeks elapsed between
experiments (crossover fashion). Blood samples (6 ml) used to determine drugconcentrations were obtained 5, 10, 15, 30, 60, 120, and 240 min afteradministration of furosemide. The blood was immediately centrifuged at 3000
rpm for 10 min, and the serum was stored at ⫺80°C until analysis for

r⫽

关D b兴
⫽
关Pt兴

冘
j

i⫽1

niKi关Df兴
1 ⫹ Ki关Df兴(1)where r is the number of moles of bound drug per protein molecule, [Db] and[Df] are the bound and unbound drug concentrations, [Pt] is the total protein
concentration, and Ki and ni are the binding constant and the number of bindingsites, respectively, for the class of binding sites. The simultaneous binding of
two ligands was analyzed using a previously reported method (Kragh-Hansen,
1981), as follows:rA ⫽KA关Af兴 ⫹ ␹KBAKB关Af兴关Bf兴关Ab兴⫽
关Pt兴 1 ⫹ KA关Af兴 ⫹ KB关Bf兴 ⫹ ␹KBAKB关Af兴关Bf兴

(2)

rB ⫽

KB关Bf兴 ⫹ ␹KABKA关Af兴关Bf兴关Bb兴⫽
关Pt兴 1 ⫹ KA关Af兴 ⫹ KB关Bf兴 ⫹ ␹KBAKB关Af兴关Bf兴

(3)

where KA and KB are the binding constants of ligands A and B, [Af] and [Bf]are the free concentrations of ligands A and B, and [Ab] and [Bb] are the boundconcentrations of ligands A and B. ␹ is a coupling constant, KBA is the binding
constant of ligand A in the presence of ligand B, and KAB is the binding
constant of ligand B in the presence of ligand A. Using these equations, we
calculated the theoretical values of ␹. The interaction mode of the ligands on
a macromolecule can be evaluated from the sign and magnitude of the value of␹. For example, if ligands A and B are independently bound to protein, ␹ isequal to 1. The results ␹ ⬎ 1 and 0 ⬍ ␹ ⬍ 1 indicate cooperative andanti-cooperative interaction between ligands. Competitive displacement between ligands is indicated by ␹ ⫽ 0. In this analysis, r ⬍ 0.42 was used tosuppress the contribution of a low-affinity binding site.
Inhibition of furosemide binding by bucolome was estimated from monitored changes of the free ligand fraction, fu, which was calculated as follows:
fu ⫽

关Df兴
关Df兴 ⫹ 关Db兴

(4)

Pharmacokinetics of Furosemide in Normal and Nephrotic Syndrome
Model Rats. Adult male Wistar rats were housed in an air-conditioned roomwith free access to commercial feed and water, and fasted for 16 h before theabdominal aorta injection. All animal experiments were conducted according
to the guideline principle and procedures of Kumamoto University for the careand use of laboratory animals. Experimental nephrotic syndrome was induced
in rats by administration of Adriamycin in a single injection (7.5 mg/kg) via thetail vein (referred to as nephrotic syndrome model rats, or NS rats) (Bertani etal., 1982). Control rats were injected with isotonic saline. Male Wistar rats
(250 –290 g; 2 weeks after i.v. administration of Adriamycin or saline) underwent the surgical procedure under light anesthesia with diethyl ether. A
cannula was inserted into the femoral vein and artery, using polyethylene
tubing (polyethylene-50; i.d., 0.58 mm; o.d., 0.9655 mm; BD Biosciences, San

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serum albumin (HSA), which is one of the major binding sites for
drugs on HSA. Also, we found that valproic acid, phenytoin, andbucolome inhibited binding of furosemide to HSA (Takamura et al.,1996). Bucolome, a nonsteroidal anti-inflammatory drug, can be administered in large doses (600 –1200 mg/day); its plasma concentration reaches about 300 ␮M, and it is excreted mainly in urine (Kakemi
et al., 1970; Yashiki et al., 1971a,b; Chiba et al., 1985).
In the present study, we investigated the effects of bucolome on thepharmacokinetics and pharmacodynamics of furosemide in healthy
subjects and doxorubicin (Adriamycin)-induced nephrotic syndromemodel rats. First, we studied the effects of bucolome on protein
binding of furosemide in serum and urine, and found that bucolome isan effective binding inhibitor for furosemide. Moreover, we foundthat coadministration of bucolome affects the pharmacokinetics of
furosemide, based on the inhibition of protein binding, suggesting an
increased natriuretic effect of the diuretic. These results suggest thatbucolome can alleviate diuretic resistance in patients with nephrotic
syndrome.

598

TAKAMURA ET AL.

Jose, CA). The bladder was also cannulated with polyethylene tubing (polyethylene-8; o.d., 2.33 mm; Hibiki Co., Tokyo, Japan.). The body temperatureof the rats was maintained by heating with a lamp. Thirty minutes before thei.v. injection of furosemide, control samples of urine were collected. Furosemide was administered at a dose of 2 mg/kg by rapid infusion into thefemoral vein, alone or 1 h after oral pretreatment with bucolome considering
the pharmacokinetics of bucolome (tmax of bucolome in rats ⫽ 53.5 min, data
not shown). After infusion, the cannulae were flushed with a small volume ofheparinized saline, to ensure that the complete dose was administered and toprevent the formation of clots. Blood samples (200 ␮l) and urine were takenfrom the femoral artery and bladder, respectively, at designated times. The
blood was placed in graduated microcentrifuge tubes (0.6 ml) that contained adrop of heparinized saline as an anticoagulant. The blood samples were
centrifuged (1500g for 10 min), and the plasma was removed.
Plasma concentration profiles were analyzed by fitting the following biexponential equation with the nonlinear least-squares method (MULTI):C p ⫽ A 䡠 exp共⫺␣ 䡠 t兲 ⫹ B 䡠 exp共⫺␤ 䡠 t兲

(5)Pharmacokinetic parameters were calculated using the following equations:
AUC03⬁ ⫽ A/␣ ⫹ B/␤

(6)

CLtot ⫽ Dose/AUC(7)

t 1/2␤ ⫽ 0.693/␤

(8)

where AUC0-⬁, CLtot, and t1/2␤ represent AUC from zero to infinity, total bodyclearance, and half-life of the ␤ phase, respectively.
Statistical Analysis. Student’s t test was used to analyze differences between two groups. Analysis of variance was used to analyze differences among
more than two groups, and the significance of difference between two meansin these groups was evaluated using the modified Fisher’s least-squares difference method.

Results
Effect of Bucolome on the Protein Binding of furosemide In
Vitro. To estimate the effect of bucolome on the protein binding of
furosemide, we have measured the free fraction of furosemide in
human serum in the absence and presence of bucolome. Protein
binding of furosemide (20 ␮M) was significantly inhibited by 300 ␮Mbucolome, and fu of furosemide in the presence of bucolome (fu ⫽3.00 ⫾ 0.12%) was approximately 3-fold greater than the value in theabsence of bucolome (fu ⫽ 1.10 ⫾ 0.09%).
Figure 1 shows the results of a quantitative analysis of mutual

displacement between furosemide and bucolome. The kinetic binding
constant (n1 䡠 K1) of furosemide on the binding to HSA was reported
to be 2.0 ⫻ 105 M⫺1 (Takamura et al., 1996), and that of bucolome
was determined to be 1.5 ⫻ 106 M⫺1 (data not shown). Taking theirparameters into consideration, a 60 ␮M concentration of these ligands
will inhibit the binding to albumin. In this condition, the binding
isotherm of furosemide in the presence of bucolome, and vice versa,
was fairly close to the theoretical curve that assumed competition
between these two ligands at a common site.
Effect of Bucolome on the Pharmacokinetics of Furosemide in
Healthy Volunteers. Table 1 summarizes the pharmacokinetic parameters of furosemide after intravenous administration (20 mg) withand without oral coadministration of bucolome (600 mg) in healthy
volunteers. In the group with coadministration of bucolome, AUC
decreased significantly, and CLtot, renal clearance (CLr), and steady-state
volume of distribution (Vdss) increased significantly. However, t1/2␤ was unchanged.
These results suggest that the alteration of pharmacokinetics of furosemide wascaused by inhibition of protein binding by bucolome. In fact, at 5, 10, and 15 minafter administration of furosemide, the free fraction of furosemide increased significantly in the group with coadministration of bucolome (Fig. 2).

Effect of Bucolome on the Natriuretic Effect of Furosemide in
Healthy Volunteers. To evaluate the pharmacodynamics of furosemide, urine volume and sodium concentration in urine were measured at 6 h after intravenous administration of furosemide. As mentioned above, in the group with coadministration of bucolome, renal
clearance of furosemide was increased (Table 1). This result is consistent with the significant increase of urine volume and sodium
concentration in urine (Table 2).
Effect of Bucolome on the Protein Binding of Furosemide in
Urine. To restore the diuretic response in patients with nephrotic
syndrome, a binding inhibitor (bucolome) must displace the diuretic(furosemide) in urine containing a high concentration of albumin. To
determine whether bucolome inhibits the protein binding of furosemide in urine, ultrafiltration was performed using artificial nephrotic urine, which contains a level of albumin equivalent to that ofpatients with nephrotic syndrome. The results indicate that bucolome
inhibited the protein binding of furosemide in the artificial nephrotic
urine (Fig. 3A). This inhibition was not affected by changes in the pHof urine that simulated the acidosis that occurs in nephrotic syndrome(Fig. 3B).

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FIG. 1. Binding of furosemide in the presence of bucolome (A) and vice versa (B) to HSA at pH 7.4 and 25°C. A, binding of furosemide (10 –20 ␮M) to HSA (120 ␮M)
in the presence of bucolome (60 ␮M). B, binding of bucolome (30 –50 ␮M) to HSA (120 ␮M) in the presence of furosemide (60 ␮M). Circles, experimental values; dashedline; theoretical curve assuming competitive binding; solid line, theoretical curve assuming independent binding. All theoretical curves were constructed using the n1 andK1 values [furosemide, n1 䡠 K1 ⫽ 2 ⫻ 105 M⫺1 (Takamura et al., 1996); bucolome, n1 䡠 K1 ⫽ 1.5 ⫻ 106 M⫺1].

BUCOLOME ADVANCES THE DIURETIC EFFECT OF FUROSEMIDETABLE 1
Kinetic parameters of furosemide after single intravenous injection of furosemide
to healthy volunteers alone (A) and with bucolome (B)Each value represents the mean ⫾ S.D. (n ⫽ 3).

AUC (mg/l 䡠 h)
CLtot (l/h)
CLr (l/h)
Vdss (liters)
t1/2␤ (h)Furosemide
Alone

With Bucolome

2.55 ⫾ 0.22
7.89 ⫾ 0.60
3.63 ⫾ 0.57
5.54 ⫾ 0.96
0.67 ⫾ 0.28

1.84 ⫾ 0.24***
11.0 ⫾ 1.35*
8.37 ⫾ 1.43**
8.57 ⫾ 1.11**
0.69 ⫾ 0.26

* p ⬍ 0.05; ** p ⬍ 0.01; *** p ⬍ 0.001 vs. furosemide alone.

599

Discussion

FIG. 2. Serum protein binding of furosemide after single intravenous injection of
furosemide to healthy volunteers, alone (A) and with bucolome (B). The serum-freefraction of furosemide was determined at 5, 10, and 15 min after single intravenous
administration. Each column is the mean of three experiments ⫾ S.D. 多, p ⬍ 0.05
(A versus B); 多多, p ⬍ 0.01 (A versus B). Statistical analysis was performed bypaired t test.
TABLE 2Urine volume and urinary excretion of sodium for 6 h after single intravenous
injection of furosemide to healthy volunteers alone (A) and with bucolome (B)Each value represents the mean ⫾ S.D. (n ⫽ 3).

Urine volume (liters)
Urinary sodium (mM)

Furosemide Alone

With Bucolome

1.23 ⫾ 0.15
7.89 ⫾ 0.60

1.48 ⫾ 0.12*
11.0 ⫾ 1.35** p ⬍ 0.05 vs. furosemide alone.

Effect of Bucolome on the Pharmacokinetics of Furosemide inNormal Rats. Table 3 summarizes the pharmacokinetic parameters of
furosemide after intravenous administration (2 mg/kg) with and without oral coadministration of bucolome in normal rats. An increase in
the dose of bucolome caused a significant decrease of AUC, and asignificant increase of CLtot, CLr, Vdss, and fu.
To confirm whether furosemide and bucolome share the same

Attenuated diuretic response is frequently observed in cases ofchronic administration of furosemide (Green and Mirkin, 1980; Keller
et al., 1982; Smith et al., 1985; Kirchner et al., 1990, 1992; Wilcox,
2002). It is thought that a possible cause of this attenuation is adecrease of circulating blood volume and glomerular filtration rate,
which leads to a decrease in the concentration of sodium ions in the
thick ascending limb of Henle’s loop and a compensating increase of
sodium reabsorption in the distal tubule (Loon et al., 1989). However,in nephrotic syndrome, because a great amount of albumin is present
in urine, furosemide may bind to albumin rather than to the Na⫹-K⫹2Cl⫺ cotransporter, decreasing its effectiveness (Green and Mirkin,
1980; Keller et al., 1982; Smith et al., 1985; Kirchner et al., 1990,
1992; Wilcox, 2002). This suggests that diuretic resistance can berestored by effective inhibition of furosemide-albumin binding, whichshould cause an increase of the free concentration of furosemide inrenal tubules. The binding inhibitor should possess the following
properties: 1) it is a potent inhibitor of the protein binding of furosemide in urine; 2) when administered in large doses, its plasma
concentration reaches high levels; 3) it is primarily excreted in urine;and 4) it is highly safe and suitable for prolonged administration. In aprevious study, we demonstrated that valproic acid, phenytoin, andbucolome, which all bind to site I, inhibited the protein binding of
furosemide when administered at typical clinical doses (Takamura etal., 1996). Bucolome, a nonsteroidal anti-inflammatory drug, is usually administered in large doses (600 –1200 mg); its plasma concentration is typically about 300 ␮M, and it is primarily eliminated byurine excretion (Kakemi et al., 1970; Yashiki et al., 1971a,b; Chiba etal., 1985). It has been reported that bucolome does not affect urinary
enzyme activities, suggesting that any nephrotoxicity of bucolome
would be very low-level (Tsurumi et al., 1978). Also, bucolome has
been used to treat nephrotic syndrome patients in warfarin-bucolome
combination therapy (Sato et al., 1991). Thus, it appears that buco-

Downloaded from dmd.aspetjournals.org at Kanazawa Daigaku Igakubu on July 10, 2009binding site on RSA, we studied the interaction between furosemide
and bucolome. In vitro binding analysis indicated that bucolome
inhibited the binding of furosemide competitively (data not shown).
Furosemide and bucolome have approximately the same affinity for
RSA (furosemide, Ka ⫽ 4.30 ⫻ 106 ⫾ 0.64 ⫻ 106 M⫺1; bucolome, Ka⫽ 6.63 ⫻ 106 ⫾ 1.79 ⫻ 106 M⫺1). The affinity of furosemide for
RSA was greater than its affinity for HSA [primary binding affinity
K1 ⫽ 2 ⫻ 105 M⫺1 (Takamura et al., 1996)], whereas bucolome had
approximately equal affinity for RSA and HSA (K1 ⫽ 1.5 ⫻ 106
M⫺1). The strong agreement between these findings and the pharmacokinetics and binding mode of furosemide in human and rats suggests that the present NS rats constitute a relevant model for analysis
of interaction between furosemide and bucolome.
Effect of Bucolome on the Diuretic Effect of Furosemide inNormal and NS Rats. Adriamycin-treated rats exhibited symptomsof nephrotic syndrome. In NS rats, serum albumin was significantly
decreased, and urinary albumin was markedly increased (Table 4).
Body weight was significantly decreased in NS rats, and hematocrit
and urine volume tended to be lower in NS rats than in normal rats.
To evaluate the effect of bucolome on diuretic resistance in NS rats,urine volume and furosemide excretion into urine were measured.
Urine volume increased with increasing dose of bucolome, which isconsistent with the significant increase of urinary excretion of furosemide in normal rats (Fig. 4, A and C). Although NS rats exhibited
attenuation of diuretic response, compared with normal rats (Fig. 4, C
and D), diuretic resistance was restored by oral coadministration of
bucolome, accompanied by an increase of urinary excretion of furosemide (Fig. 4, B and D).

600

TAKAMURA ET AL.

TABLE 3
Pharmacokinetic parameters of furosemide after single intravenous injection to normal rats (2 mg/kg) alone and with oral coadministration of bucolome
Each value represents the mean ⫾ S.D. (n ⫽ 3– 4).
With Bucolome

Furosemide
Alone

Parameter

AUC (␮g/ml 䡠 min)CLtot (ml/min/kg)
CLr (ml/min/kg)
Vdss (ml/kg)
t1/2␤ (min)fb (%)

563 ⫾ 82
3.61 ⫾ 0.52
1.08 ⫾ 0.16
75.7 ⫾ 6.0
44.7 ⫾ 12.7
1.28 ⫾ 0.36

10 mg/kg

15 mg/kg

20 mg/kg572 ⫾ 31
3.51 ⫾ 0.19
1.42 ⫾ 0.25
75.1 ⫾ 1.3
33.6 ⫾ 3.7
1.41 ⫾ 0.72

414 ⫾ 47*
4.87 ⫾ 0.52
2.11 ⫾ 0.21**
92.1 ⫾ 5.6**
35.7 ⫾ 9.6
1.99 ⫾ 0.76

336 ⫾ 91**
6.24 ⫾ 1.54*
2.67 ⫾ 0.42**
112 ⫾ 7**29.4 ⫾ 4.5
3.03 ⫾ 0.57*** p ⬍ 0.05; ** p ⬍ 0.01 vs. furosemide alone.

TABLE 4
Biochemical parameters at 2 weeks after intravenous administration of
Adriamycin (9 mg/kg)
Each value represents the mean ⫾ S.D. (n ⫽ 3– 6).

Body weight (g)Hematocrit (%)
Serum albumin level (ml/min/kg)
Urine volume (ml/day)Proteinuria (mg/day)ControlWith Adriamycin

203 ⫾ 1137.4 ⫾ 3.9
3.75 ⫾ 0.14
6.53 ⫾ 0.47
9.04 ⫾ 4.15

176 ⫾ 16*35.4 ⫾ 1.7
3.03 ⫾ 0.23**
6.27 ⫾ 3.00
275 ⫾ 84*

* p ⬍ 0.05; ** p ⬍ 0.01 vs. control.

lome is suitable for treatment of nephrotic syndrome patients, despite
the need for careful monitoring of renal function.
The diuretic resistance caused by decreased levels of the active
form (i.e., unbound form) of furosemide in urine can be alleviated byinhibiting protein binding of furosemide, which increases the amountof free furosemide delivered to the site of action. In the present study,
when bucolome was administered with furosemide in healthy volun-teers, the free fraction of furosemide in plasma increased (Fig. 2), as
predicted by the in vitro data (Fig. 1). The free fractions of someligands were slightly influenced by the temperature (Melten et al.,1986), suggesting that the present protein binding obtained by ultrafiltration at 25°C might be greater than that at 37°C. However, interms of the qualitative analysis, it is suggested that bucolome inhibits
the binding of furosemide under in vivo physiological conditions.
Therefore, a decrease in the AUC and an increase in the CLtot and Vdss
would be due to the decrease in the protein binding of furosemide(Table 1). Also, the natriuretic effect of furosemide was reinforced bycoadministration of bucolome (Table 2). We hypothesize that thealterations of these pharmacokinetic parameters were caused by theincrease in the amount of furosemide available for the hepatic metabolism and renal excretion, followed by the increase in the free fractionof furosemide. The increase of renal clearance of furosemide resulted
in the increase of the amount reaching the site of action (Table 1), theNa⫹-K⫹-2Cl⫺ cotransporter and induced the acceleration of the diuretic effect (Table 2).
Even though the amount of furosemide reaching the site of action

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FIG. 3. Effect of bucolome on the protein binding of furosemide in UPA. A, concentration-dependent protein binding of furosemide (30 ␮M, bars A and B; 60 ␮M, bars
C and D) was examined without bucolome (bars A and C) and with 500 ␮M bucolome (bars B and D) at 25°C. Concentration of urinary protein was 480 ␮M (as HSA).
B, the effect of pH on the protein binding of furosemide was examined in the presence of bucolome in UPA at 25°C. The following concentrations were used: [urinary
protein], 480 ␮M (as HSA); [furosemide], 30 ␮M; [bucolome], 500 ␮M. Each bar represents mean ⫾ S.D. (n ⫽ 3). 多, p ⬍ 0.001 versus furosemide alone.

BUCOLOME ADVANCES THE DIURETIC EFFECT OF FUROSEMIDE601

was increased by the coadministration of bucolome, unless the protein
binding of furosemide in urine is inhibited, the blunted response to
furosemide may not be alleviated in patients with nephrotic syndrome.
In the present study, accurate concentrations of drugs tested in thenephrotic loop could not be estimated. However, considering thepharmacokinetic parameters of furosemide (Table 1) and bucolome(Yashiki et al., 1971a,b), their urinary concentrations were expected toreach about 30 and 500 ␮M, respectively. Additionally, the clinicaldose of furosemide is often increased in patients with nephrotic
syndrome. Therefore, we assumed that urinary concentrations of furosemide and bucolome in the renal tubules were 30 or 60 ␮M, and500 ␮M, respectively (Fig. 3A), although they would be more concentrated than the artificial urine used in this experiment. As a result,
we demonstrated that bucolome effectively inhibited the protein binding of furosemide, even in urine. Sulfamethizole and sulfisoxazole,which bind to site I of HSA, did not inhibit the protein binding of
furosemide in urine, even at doses of 1 mM, although they inhibit
binding effectively in plasma (data not shown). This suggests that it isimportant for the binding inhibitor to possess the high affinity foralbumin exhibited by bucolome (n1 䡠 K1 ⫽ 1.5 ⫻ 106 M⫺1). In
addition, as an inhibitor for protein binding of furosemide in urine,

bucolome has the advantage that its effects are not altered by changes
in pH (Fig. 3B) or the presence of other drugs, including sulfamethizole and sulfisoxazole. The binding percentage of furosemide in
artificial urine was about 70% (Fig. 3A), which was lower than thepercentage in plasma (about 99%), suggesting that alteration of pH
between urine and plasma, and/or endogenous inhibitors, such as fatty
acid, uremic toxins, and eicosanoids, may be involved in the protein
binding of furosemide in the urine (Kragh-Hansen et al., 2002).
Interestingly, we found that coadministration of bucolome with
furosemide in Adriamycin-induced NS rats alleviated the diuretic
resistance (Fig. 4). It has been repeatedly shown that the urinary
concentration of loop diuretic is the best index of drug concentration
at the intraluminal site of action in the thick ascending limb of Henle’s
loop. Also, there are publications for furosemide that depict urine
output versus excretion rates to better reflect the pharmacodynamics(Green and Mirkin, 1980; Keller et al., 1982; Smith et al., 1985;
Kirchner et al., 1990, 1992; Wilcox, 2002). In the present study,
promotion of diuretic effects by bucolome was accompanied by anincreasing urinary excretion rate of furosemide (Fig. 4), suggesting an
increase in the amount of furosemide delivered to the site of action.
These results are in good agreement with the present findings for

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FIG. 4. Urinary excretion of furosemide (A and B) and urine volume (C and D) after i.v. administration of furosemide to normal (A and C) and Adriamycin-treated rats
(B and D) alone and with bucolome. Bucolome was orally coadministered (E, 0 mg/kg; F, 10 mg/kg; Œ, 15 mg/kg; f, 20 mg/kg) with a single intravenous injection of
furosemide (2 mg/kg) to normal and Adriamycin-treated rats. Each point represents the mean ⫾ S.D. (n ⫽ 4 –9). 多, p ⬍ 0.05; 多多, p ⬍ 0.01 versus furosemide alone.

602

TAKAMURA ET AL.

References
Agarwal R, Gorski JC, Sundblad K, and Brater DC (2000) Urinary protein binding does notaffect response to furosemide in patients with nephrotic syndrome. J Am Soc Nephrol
11:1100 –1105.
Bertani T, Poggi A, Pozzoni R, Delaini F, Sacchi G, Thoua Y, Mecca G, Remuzzi G, and Donati
MB (1982) Adriamycin-induced nephrotic syndrome in rats: sequence of pathologic events.
Lab Investig 46:16 –23.
Chiba K, Yoshida M, Ishihara A, Sudo J, Tanabe T, Miyazaki S, and Takada M (1985) Study ofinteractions between ibuprofen and sulfonamides and between bucolome and sulfonamides inrats. Chem Pharm Bull (Tokyo) 33:3415–3421.
Green TP and Mirkin BL (1980) Resistance of proteinuric rats to furosemide: urinary drug
protein binding as a determinant of drug effect. Life Sci 26:623– 630.
Kakemi K, Sezaki H, Komuro T, Ikeda K, and Kishi H (1970) Effect of 5-n-butyl-1-cyclohexyl2,4,6-trioxoperhydropyrimidine on the sulfonamides transfer in the rat. Chem Pharm Bull
(Tokyo) 18:2386 –2392.
Keller E, Hoppe-Seyler G, and Schollmeyer P (1982) Disposition and diuretic effect of furosemide in the nephrotic syndrome. Clin Pharmacol Ther 32:442– 449.
Kirchner KA, Voelker JR, and Brater DC (1990) Intratubular albumin blunts the response to

furosemide—a mechanism for diuretic resistance in the nephrotic syndrome. J Pharmacol Exp
Ther 252:1097–1101.
Kirchner KA, Voelker JR, and Brater DC (1991) Binding inhibitors restore furosemide potency
in tubule fluid containing albumin. Kidney Int 40:418 – 424.
Kirchner KA, Voelker JR, and Brater DC (1992) Tubular resistance to furosemide contributes tothe attenuated diuretic response in nephrotic rats. J Am Soc Nephrol 2:1201–1207.
Kragh-Hansen U (1981) Molecular aspects of ligand binding to serum albumin. Pharmacol Rev
33:17–53.
Kragh-Hansen U, Chuang VT, and Otagiri M (2002) Practical aspects of the ligand-binding andenzymatic properties of human serum albumin. Biol Pharm Bull 25:695–704.
Loon NR, Wilcox CS, and Unwin RJ (1989) Mechanism of impaired natriuretic response to
furosemide during prolonged therapy. Kidney Int 36:682– 689.
Maes V, Engelborghs Y, Hoebeke J, Maras Y, and Vercruysse A (1982) Fluorimetric analysis of
the binding of warfarin to human serum albumin. Equilibrium and kinetic study. Mol
Pharmacol 21:100 –107.
Martin BK (1965) Potential effect of the plasma on drug distribution. Nature (Lond) 207:274 –276.
Melten JW, Wittebrood AJ, Wemer J, and Faber DB (1986) On the modulating effects oftemperature, albumin, pH and calcium on the free fractions of phenobarbitone and phenytoin.
J Pharm Pharmacol 38:643– 646.
Meyer MC and Guttman DE (1968) The binding of drugs by plasma proteins. J Pharm Sci
57:895–918.
Sato H, Tawaraya H, Takagi A, Wakabayashi M, Yoshida K, Ueno M, Maruyama Y, Suzuki S,
and Arakawa M (1991) [A case of heterozygous protein C deficiency associated with nephrotic
syndrome and deep femoral artery thrombosis]. Nippon Jinzo Gakkai Shi 33:327–332.
Smith DE, Hyneck ML, Berardi RR, and Port FK (1985) Urinary protein binding, kinetics anddynamics of furosemide in nephrotic patients. J Pharm Sci 74:603– 607.
Takamura N, Haruta A, Kodama H, Tsuruoka M, Yamasaki K, Suenaga A, and Otagiri M (1996)
Mode of interaction of loop diuretics with human serum albumin and characterization ofbinding site. Pharm Res (NY) 13:1015–1019.
Takamura N, Maruyama T, and Otagiri M (1997) Effects of uremic toxins and fatty acids onserum protein binding of furosemide: possible mechanism of the binding defect in uremia. Clin
Chem 43:2274 –2280.
Takamura N, Shinozawa S, Maruyama T, Suenaga A, and Otagiri M (1998) Effects of fatty acids
on serum binding between furosemide and valproic acid. Biol Pharm Bull 21:174 –176.
Tsurumi K, Abe A, Nozaki M, and Fujimura H (1978) Influence of some anti-inflammatory,
antipyretic and analgesic agents on urinary enzyme level in rats. J Toxicol Sci 3:1–9.
Tsutsumi Y, Maruyama T, Takadate A, Goto M, Matsunaga H, and Otagiri M (1999) Interaction
between two dicarboxylate endogenous substances, bilirubin and an uremic toxin, 3-carboxy4-methyl-5-propyl-2-furanpropanoic acid, on human serum albumin. Pharm Res (NY) 16:916 –923.
Vallner JJ (1977) Binding of drugs by albumin and plasma protein. J Pharm Sci 66:447– 465.
Wilcox CS (2002) New insights into diuretic use in patients with chronic renal disease. J Am Soc
Nephrol 13:798 – 805.
Yamaoka K, Tanigawara Y, Nakagawa T, and Uno T (1981) A pharmacokinetic analysis
program (MULTI) for microcomputer. J Pharmacobio-Dyn 4:879 – 885.
Yashiki T, Matsuzawa T, Yamada M, Kondo T and Mima H (1971a) Studies on the metabolic
fate and the pharmacokinetics of 5-n-butyl-1-cyclohexyl-2,4,6-trioxoperhydropyrimidine(BCP) in man. V. Pharmacokinetics of BCP in man and in rabbit following intravenous
administration of BCPNa. Chem Pharm Bull (Tokyo) 19:881– 891.
Yashiki T, Matsuzawa T, Yamada M, Kondo T, and Uda Y (1971b) Studies on the metabolic fate
and the pharmacokinetics of 5-n-butyl-1-cyclohexyl-2,4,6-trioxoperhydropyrimidine (BCP) inman. IV. Pharmacokinetics of BCP in man following oral administration. Chem Pharm Bull
(Tokyo) 19:869 – 880.

Address correspondence to: Professor Masaki Otagiri, Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan. E-mail: otagirim@gpo.
kumamoto-u.ac.jp

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healthy subjects, suggesting that this therapeutic strategy can restore
the diuretic response to furosemide in patients with nephrotic syndrome. The affinity of furosemide for RSA was higher than its affinity
for HSA, although bucolome had approximately equal affinity for
RSA and HSA. The inhibitory effect of bucolome on the protein
binding of furosemide in plasma and urine is likely to be stronger inhumans than rats, suggesting that bucolome has a greater effect on thediuretic properties of furosemide in patients with nephrotic syndrome
than in NS rats. On the other hand, the mean urine volume attained by
the coadministration of bucolome (20 mg/kg) with furosemide in the
NS rats (Fig. 4D, 395–753 ␮l) was still less than that observed innormal rats (Fig. 4C, 1352–2103 ␮l), although coadministration of
bucolome with furosemide doubled the urinary excretion of furosemide at 3 h postdose (Fig. 4, A and B). Thus, the present limited
data suggested that coadministration of bucolome with furosemide
may partially restore the diuretic resistance in the nephrotic rats. A
critical element of the present strategy is to increase the amount of
free furosemide delivered to the site of action. In the condition of
repeated administration, that is, the clinically relevant situation, highconcentrations of furosemide and bucolome would be maintained in
the urine. Therefore, the inhibitory effect of bucolome by chronic
dosing may be more possibly generated compared with that by single
dosing.
The present results suggest that coadministration of bucolome canpartially reverse the diuretic resistance of furosemide in patients withnephrotic syndrome. These findings may lead to an effective clinicaltherapy for alleviation of diuretic resistance of furosemide in nephrotic syndrome.